Novel cyanoenamines useful as ligands for modulating gene expression in plants or animals

ABSTRACT

Accordingly, the present invention provides a compound of Formula I, or tautomers or isomers thereof,  
                 
 
     wherein: R1 is a branched chain lower alkyl (C3 to C8), cycloalkyl (C3 to C8), alkyl-substituted alkyl (C4 to C8), bicycloalkyl, 1-adamantyl, polyhaloalkyl, trialkylsilyl, unsubstituted phenyl or substituted phenyl; R2 and R3 are independently unsubstituted or substituted aromatic rings; and R4 is hydrogen, alkyl, alkylthio, alkylthioalkyl, acyloxyalkyl, alkyl, acyl, trialkylsilyl, or cyclized together with R3 and O to form a lactone of the tautomeric form.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present patent application claims benefit of U.S. Provisional Patent Application Serial No. 60/272,905, filed Mar. 2, 2001 and is incorporated herein by reference.

TECHNICAL FIELD

[0002] The present invention relates, in general, to novel compounds that are useful as ligands for modulating gene expression in living organisms (plants and/or animals). More particularly, the present invention relates to compounds that are cyanoenamines that are useful as non-steroidal ligands for modulating exogenous gene expression in eukaryotic organisms (i.e., those where the cell has a nucleus), more particularly plants, especially chlorophyll-containing plants. Table of Abbreviations n-BuLi n-butyllithium t-butyl tert-butyl C centigrade DMAP N,N-dimethylaminopyridine DNA deoxyribonucleic acid EcR ecdysone receptor EC80 effective concentration that produces an 80% effect GC gas chromatography GR glucocorticoid receptor g gram Hv Heliothis virescens h hour L1 first larval instar, namely the stage between the egg and the first molt LC liquid crystal LDA lithium diisopropylamide MS mass spectroscopy mp melting point μM micromole mL milliliter mm millimeter min minute M mole NMR nuclear magnetic resonance # number ppm parts per million RNA ribonucleic acid RXR retinoid X receptor SPODLI Spodoptera littoralis THF tetrahydrofuran USP ultraspiracle

BACKGROUND OF THE INVENTION

[0003] Precise temporal control of gene expression is a valuable tool in the field of genetic engineering. The ability to activate (i.e., to induce) or to suppress a gene is of vast importance in manipulating, controlling, and/or studying development and other physiological processes. Inducability is often valuable for foreign protein production, such as production of therapeutic proteins, industrial enzymes, and polymers, in both plants and animals.

[0004] Specifically in the case of plants, often desirable is the control of the timing and level of expression of a phenotypic trait in a plant, plant cell or plant tissue. Ideally, regulation of expression of such a trait can be achieved whenever desired by triggering gene expression with a chemical that is easily applied to field crops, ornamental shrubs and other plants of economic importance. This triggering mechanism for gene expression control is referred to as a gene switch. In order to avoid unexpected activation of the gene switch, the chemical should be one that is normally absent from the plant.

[0005] One such gene switch mechanism is the ecdysone receptor (EcR). EcR is a member of the nuclear hormone family of receptors. Members of this receptor family are multi-domain proteins, capable of regulating gene expression in response to a chemical ligand. The DNA binding domain (also known as the C domain) binds to a specific target DNA sequence. This specificity determines which target genes are activated by the receptor. The ligand binding domain (E domain) plays a critical role in the determination of ligand specificity as well as the ligand regulated activation property of the receptor. The hinge domain (domain D) resides between the DNA binding and ligand binding domains. The hinge domain modulates the receptor's response to ligand induction.

[0006] Ligands that are complementary to the ligand binding domain of the ecdysone receptor are known. Steroidal agonists such as 20-hydroxy ecdysone, muristerone, and ponasterone are capable of activating an ecdysone receptor gene switch. Non-steroidal agonists have advantages over steroidal agonists due to such factors as greater stability, cheaper cost, and environmental acceptance. One known non-steroidal agonist is the insecticide Tebufenozide (also known as the insecticide sold under the trademark MIMIC®).

[0007] Of interest is European Published Patent Application No. 0 965 644 A2 to Carlson et al., assignors to Rohm and Haas Company, which relates to a method of modulating exogenous gene expression in which an ecdysone receptor complex is contacted with a DNA construct having an exogenous gene under the control of a response element, and where the binding of the ecdysone receptor to the response element results in activation or suppression of the gene. The ligand is chosen from certain dibenzoyl-tert-butyl-hydrazine compounds.

[0008] As referred to herein, an “ecdysone receptor gene switch” means a gene switch comprising an ecdysone receptor. The ecdysone receptor gene switch may be a heterodimer of EcR and USP, or EcR and RXR. The heterodimerization partner may be native to the organism or cell type in which the gene switch is present, or the heterodimerization partner may be provided exogenously. The ecdysone receptor gene switch may be comprised only of EcR in the absence of a heterodimerization partner. EcR may be in its native form, as isolated from insects, comprising a DNA binding domain, hinge and ligand binding domain from an insect EcR. EcR may be a chimeric protein comprising a DNA binding domain from another EcR or another transcription factor such as Ga14. EcR may comprise its native activation domain or an activation domain of another protein. Furthermore, EcR may comprise a ligand binding domain of an insect ecdysone receptor or a ligand binding domain from a member of the nuclear hormone family of receptors.

[0009] Also of interest is International Publication No. WO 00/15791 to Albertsen et al., assignors to Pioneer Hi-Bred International, Inc. This Publication relates to novel ecdysone receptors from the insect species Ostrinia and the genus Pyrilidae and their use for gene regulation in plants.

[0010] Additionally of interest is International Publication No. WO 99/02683 to Gage et al., assignors to The Salk Institute for Biological Studies. This Publication relates to nuclear receptor proteins from the silk moth Bombyx mori, useful for the regulation of gene expression.

[0011] Also of interest is International Publication No. WO 96/37609 to Jepson et al., assignors to Zeneca, relating to the use of a chimeric ecdysone receptor gene switch in plants.

[0012] Of general background interest is each of the following describing examples of ecdysone receptor gene switches: No et al., Proc. Nat'l. Acad. Sci., 93: 3346-3351 (1996), describing EcR in mammalian cells; Godowski et al., International Publication No. WO 93/03162, describing EcR and chimeric EcR proteins and related gene switches; Evans et al., International Publication Nos. WO 99/58155 and WO 97/38117, describing EcR and chimeric EcR proteins and related gene switches; Martinez et al., Insect. Biochem. Mol. Biol., 29 (10):915-930 (October, 1999), describing a chimeric EcR gene switch in plants; Martinez et al., Plant J., 19(1):97-106 (July, 1999), describing a chimeric EcR gene switch in plants; Martinez et al., Mol. Gen. Genet., 261(3):546-552 (April, 1999), describing a chimeric EcR gene switch in plants; Suhret al., Proc. Nat'l. Acad. Sci. U.S.A., 95(14):7999-8004 (Jul. 7, 1998), describing a chimeric EcR switch in mammalian cells; and Hoppe et al., Mol. Ther., 1(2):159-164 (February, 2000), describing an adenovirus mediated EcR gene switch.

[0013] Especially of interest is U.S. Pat. No. 5,880,333 to Goff et al., assignors to Novartis Finance Corporation. This patent discloses a method of controlling gene expression in plants. Specifically, the method involves obtaining a transgenic plant that has at least 2 receptor expression cassettes and at least 1 target expression cassette. A first of the 2 receptor expression cassettes has a nucleotide sequence for a 5′ regulatory region operably linked to a nucleotide sequence that encodes a first receptor polypeptide and a 3′ termination region. A second of the 2 receptor expression cassettes has a nucleotide sequence for a 5′ regulator region operably linked to a nucleotide sequence that encodes a second receptor polypeptide and a 3′ termination region. The target expression cassette has a nucleotide sequence operably linked to a nucleotide sequence that encodes a target polypeptide and a 3′ termination region, wherein the 5′ regulatory region of the target expression cassette is activated by the first and second receptor polypeptides in the presence of a certain chemical ligand that is complimentary to the ligand binding domain of the receptor polypeptides, as a result of which expression of the target polypeptide is accomplished. In a preferred embodiment, the method involves expressing in a plant an insect EcR and a second receptor as a heterodimerization partner and activating the expression of a target polypeptide by contacting the plant cells with a ligand that is complimentary to the ligand binding domain of one of the receptors. The method of U.S. Pat. No. 5,880,333 to Goff et al. is useful for controlling various traits of agronomic importance, such as plant fertility.

[0014] Lastly, of interest is U.S. Provisional Application No. 60/242,969, filed Oct. 24, 2000, describing novel ecdysone receptor gene switches and methods of use, the disclosure of which is incorporated in its entirety.

[0015] All of the patents and published patent applications mentioned here are incorporated by reference.

[0016] Despite the plethora of available ecdysone receptor gene switch systems, there still remain a continuing need to develop non-steroidal ligands with increased activity as compared to known ligands and a need to develop ligands that demonstrate improved consistent activity in intact plants and animals.

SUMMARY AND OBJECTS OF THE INVENTION

[0017] Accordingly, the present invention provides a compound comprising Formula I

[0018] and also, Formula I may be in its tautomeric form comprising Formula II

[0019] and also, Formula I may be in its isomeric form comprising Formula III

[0020] wherein:

[0021] R1

[0022] is a branched chain lower alkyl (C3 to C8), cycloalkyl (C3 to C8), alkyl-substituted alkyl (C4 to C8), bicycloalkyl, 1-adamantyl, polyhaloalkyl, trialkylsilyl, unsubstituted phenyl or optionally substituted phenyl;

[0023] R2 and R3

[0024] are independently unsubstituted or substituted aromatic rings, chosen from phenyl, pyridyl, pyrimidinyl, furyl, thiophenyl, pyrazinyl, pyrrolyl, pyrazolyl, 1,2,4-triazolyl, naphthyl, fluorenonyl, xanthenyl, 4-oxo-1,4-dihydro-(1,8)naphthyridinyl, thiazolyl, isothiazolyl, 1,3,4-thiadiazolyl, benzo-1,2,3-thiadiazolyl, oxazolyl, imidazolyl, quinolinyl, or isoquinolinyl, where a substituent on the rings is one or more chosen independently from hydrogen, alkyl (C1 to C4), alkoxy, alkoxyalkyl, hydroxy, amino, alkylamino, dialkylamino, acylamino, halo, haloalkyl, hydroxyalkyl, dihydroxyalkyl, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, unsubstituted or substituted alkylphenyl, unsubstituted or substituted phenyl, unsubstituted or substituted phenoxy, nitro, cyano, alkylthio, alkylsulfonyl, aminoalkyl, carboxyalkyl, or sulfonylalkyl; and

[0025] R4

[0026] is hydrogen, alkylthio, alkylthioalkyl, alkyloxyalkyl, acyloxyalkyl, alkyl, acyl, trialkylsilyl, or cyclized together with R3 and the O in Formula II to form a lactone.

[0027] The compounds described in the above paragraph are useful for modulation of an exogenous gene in a living organism. The compounds are also useful for the control of pests, such as anthropods, parasites, and the like, by acting as agonists of 20-hydroxyecdysone, the molting hormone.

[0028] Therefore, it is an object of the present invention to provide a compound that has the ability to activate or to suppress an exogenous gene.

[0029] It is another object of the present invention to provide a compound that is useful as a pesticide.

[0030] Some of the objects of the invention having been stated, other objects will become evident as the description proceeds, when taken in connection with the laboratory examples described below.

DETAILED DESCRIPTION OF THE INVENTION

[0031] The inventive ligand is an addition to the ligands described in the above-noted U.S. Pat. No. 5,880,333 to Goff et. al.

[0032] A ligand according to the present invention is described by the below recited general Formula I and its below recited tautomer, Formula II, and its below recited isomer, Formula III:

[0033] wherein:

[0034] R1

[0035] is a branched chain lower alkyl (C3 to C8), cycloalkyl (C3 to C8), alkyl-substituted alkyl (C4 to C8), bicycloalkyl, 1-adamantyl, polyhaloalkyl, trialkylsilyl, or optionally substituted phenyl;

[0036] R2 and R3

[0037] are independently optionally substituted aromatic rings, such as phenyl, pyridyl, pyrimidinyl, furyl, thiophenyl, pyrazinyl, pyrrolyl, pyrazolyl, 1,2,4-triazolyl, naphthyl, fluorenonyl, xanthenyl, 4-oxo-1,4-dihydro-(1,8)naphthyridinyl, thiazolyl, isothiazolyl, 1,3,4-thiadiazolyl, benzo-1,2,3-thiadiazolyl, oxazolyl, imidazolyl, quinolinyl, or isoquinolinyl. Substituents on these rings can be one or more chosen independently from hydrogen, alkyl (C1 to C4), alkoxy, alkoxyalkyl, hydroxy, amino, alkylamino, dialkylamino, acylamino, halo, haloalkyl, hydroxyalkyl, dihydroxyalkyl, alkoxycarbonyl, alkylaminocarbonyl, dialkyl-aminocarbonyl, (optionally substituted) alkylphenyl, (optionally substituted) phenyl, (optionally substituted) phenoxy, nitro, cyano, alkylthio, alkylsulfonyl, aminoalkyl, carboxyalkyl, and sulfonylalkyl; and

[0038] R4

[0039] is hydrogen, or a substituent that may be easily removed in planta, serving as an aid in absorption and/or translocation, such as alkylthio, alkylthioalkyl, alkyloxyalkyl, acyloxyalkyl, alkyl, acyl, trialkylsilyl, or cyclized together with R3 and the O in Formula II to form a lactone.

[0040] Halo may be selected from the group consisting of fluoro, chloro, bromo, iodo, and combinations thereof. The substituents on R2 and R3 may also be joined to form cyclic structures on adjacent atoms of the aromatic ring, such as 1,2-methylenedioxy and 1,2-difluoromethylenedioxy. The preferred R1 is tert-butyl. The preferred R2 is phenyl, 3,5-dimethylphenyl, 2,4-dimethylphenyl, 3-methylphenyl, 4-methylphenyl, 2-methylphenyl, or 3,4-methylenedioxyphenyl. The preferred R3 is phenyl, 3-pyridyl, 3-methoxy-2-methylphenyl, 3-ethoxy-2-methylphenyl, 3-methoxy-2-ethylphenyl, 4-ethylphenyl, 2,6-difluorophenyl, 2,3-dimethylphenyl, 3-chloro-2-methylphenyl, or 3-bromo-2-methylphenyl. When R3, R4, and O are cyclized to form the cyclic ester known as a lactone, the lactone may be:

[0041] The compounds described as per Formula I and its tautomer, Formula II, and its isomer, Formula III, are useful for modulation of an exogenous gene in a living organism. They have the ability to activate or to suppress an exogenous gene.

[0042] Additionally, the described compounds can be used as pesticides, i.e., for arthropod control (control of segmented invertebrates such as insects, arachnids, crustaceans, or myriapods) on plants in soil or water, in structures, and on parasites in or on vertebrate animals, acting as agonists of 20-hydroxyecdysone, the molting hormone.

[0043] The preparation of these compounds may be accomplished by the following general scheme:

[0044] and in this scheme, R1 is tert-butyl, but that is not a requirement.

LABORATORY EXAMPLES Example 1

[0045] Preparation of starting material; (E)-3-amino-2-(3,5-dimethylphenyl)-4,4-dimethylpent-2-enenitrile

[0046] To 30 mL of n-butyllithium (2.5 M in hexane) at 5° C. was added 30 mL of dry tetrahydrofuran. The resulting solution was cooled back to 5° C. and a solution of 5 g of 3,5-dimethylphenylacetonitrile in 10 mL of tetrahydrofuran was added over 30 min., keeping the temperature between 5° C. and 10° C. The mixture was stirred at 5° C. for 1 h., and then a solution of 2.86 g of trimethylacetonitrile in 10 mL of tetrahydrofuran was added. The resulting mixture was stirred overnight at ambient temperature. The mixture was poured into ice water and extracted with 2 portions of ethyl acetate.

[0047] The combined ethyl acetate layers were washed with brine, dried over MgSO₄, filtered, and evaporated in vacuo to afford the crude product as an oil, which was crystallized from petroleum ether to yield 3.2 g of a solid with a ¹HNMR spectrum consistent with the expected product, namely (E)-3-amino-2-(3,5-dimethylphenyl)-4,4-dimethylpent-2-enenitrile.

[0048] Preparation of starting material; 3-methoxy-2-methylbenzoyl chloride

[0049] Thionyl chloride (5 mL) was gradually added to 0.95 g of 3-methoxy-2-methylbenzoic acid at room temperature. The resulting mixture was heated at 65° C. for 1 h. The excess thionyl chloride was evaporated in vacuo, and a small portion of carbon tetrachloride was added. Then, the mixture was again evaporated in vacuo to yield the desired product as an oil. This was used directly in the next reaction.

[0050] Preparation of N-[(E)-1-tert-butyl-2-cyano-2-(3,5-dimethylphenyl)-vinyl]-3-methoxy-2-methylbenzamide

[0051] To 3.8 mL of lithium diisopropylamide solution (1.5 M in cyclohexane) at −78° C., was added dropwise, a solution of 0.51 g of (E)-3-amino-2-(3,5-dimethylphenyl)-4,4-dimethylpent-2-enenitrile in 30 mL of dry tetrahydrofuran. The mixture was stirred for 30 min. at −78° C., and then 1.05 g of 3-methoxy-2-methylbenzoyl chloride was added in one portion. The resulting mixture was stirred overnight at ambient temperature. The reaction mixture was poured into ice water and extracted with ethyl acetate.

[0052] The ethyl acetate layer was washed with brine, dried over MgSO₄, filtered, and evaporated in vacuo to yield 1.4 g of crude solid product. The crude product was partially purified using 3 plates, each being a 600 mm×20 mm silica gel preparative layer chromatography plate, eluted with 20% ethyl acetate in hexane to afford 0.17 g of slightly impure material. This was recrystallized from a 3 mL tetrahydrofuran and 15 mL hexane mixture to yield 0.15 g of white crystalline material (mp was 203 to 204° C.) with GC/MS and ¹HNMR spectra consistent with the desired product, namely N-[(E)-1-tert-butyl-2-cyano-2-(3,5-dimethylphenyl)-vinyl]-3-methoxy-2-methylbenzamide.

Example 2

[0053] Using essentially the same procedure as described above, the following selected cyanoenamine compounds have also been prepared, as reported in Table A1 below. TABLE A1 Melting point LC/MS Compound degrees C. molecular ion

499 Compound 1

476 Compound 2

363 Compound 3

465 Compound 4

429 Compound 5

521 Compound 6

439 Compound 7

390 Compound 8

436 Compound 9

507 Compound 10

516 Compound 11

485 Compound 12

457 Compound 13

371 Compound 14

473 Compound 15

485 Compound 16

437 Compound 17

508 Compound 18

477 Compound 19

449 Compound 20

530 Compound 21

499 Compound 22

471 Compound 23

385 Compound 24

487 Compound 25

451 Compound 26

459 Compound 27

468 Compound 28

437 Compound 29

409 Compound 30

323 Compound 31

425 Compound 32

377 Compound 33

389 Compound 34

475 Compound 35

407 Compound 36

463 Compound 37

431 Compound 38

429 Compound 39

377 Compound 40

433 Compound 41

401 Compound 42

459 Compound 43

391 Compound 44

473 Compound 45

431 Compound 46

447 Compound 47

415 Compound 48

413 Compound 49

391 Compound 50

401 Compound 51

371 Compound 52

491 Compound 53

383 Compound 54

439 Compound 55

483 Compound 56

424 Compound 57

452 Compound 58

416 Compound 59

399 Compound 60

521 Compound 61

363 Compound 62

483 Compound 63

443 Compound 64

375 Compound 65

431 Compound 66

475 Compound 67

416 Compound 68

408 Compound 69

391 Compound 70

513 Compound 71

385 Compound 72

505 Compound 73

397 Compound 74

453 Compound 75

497 Compound 76

438 Compound 77

466 Compound 78

430 Compound 79

413 Compound 80

464 Compound 81

535 Compound 82

323 Compound 83

443 Compound 84

390 Compound 85

335 Compound 86

391 Compound 87

435 Compound 88

376 Compound 89

404 Compound 90

368 Compound 91

351 Compound 92

431 Compound 93

401 Compound 94

120 Compound 95

165 Compound 96

170 Compound 97

128-133 Compound 98

165 Compound 99

195 Compound 100

145 Compound 101

190 Compound 102

150 Compound 103

198 Compound 104

153 Compound 105

205 Compound 106

130 Compound 107

165 Compound 108

165 Compound 109

82.1 Compound 110

125 Compound 111

170 Compound 112

145 Compound 113

160 Compound 114

125 Compound 115

220 Compound 116

391 Compound 117

179-182 Compound 118

175 Compound 119

170 Compound 120

225 Compound 121

184.4 Compound 122

125.9 Compound 123

171-174 Compound 124

113.6 Compound 125

>300 Compound 126

194.7 Compound 127

190 Compound 128

393 Compound 129

>300 Compound 130

178-180 Compound 131

197-200 Compound 132

203-205 Compound 133

178-180 Compound 134

173-174 Compound 135

194-195 Compound 136

194-195 Compound 137

203-204 Compound 138

146-147 Compound 139

142-144 Compound 140

175-176 Compound 141

138-140 Compound 142

180-183 Compound 143

219-222 Compound 144

[0054] The various R1, R2, R3, and R4 moieties (from the compounds made as per Table A1 above) are summarized in Table A2 below. TABLE A2 Compound # R1 R2 R3 R4 1 t-Bu

H 2 t-Bu

H 3 t-Bu

H 4 t-Bu

H 5 t-Bu

H 6 t-Bu

H 7 t-Bu

H 8 t-Bu

H 9 t-Bu

H 10 t-Bu

H 11 t-Bu

H 12 t-Bu

H 13 t-Bu

H 14 t-Bu

H 15 t-Bu

H 16 t-Bu

H 17 t-Bu

H 18 t-Bu

H 19 t-Bu

H 20 t-Bu

H 21 t-Bu

H 22 t-Bu

H 23 t-Bu

H 24 t-Bu

H 25 t-Bu

H 26 t-Bu

H 27 t-Bu

H 28 t-Bu

H 29 t-Bu

H 30 t-Bu

H 31 t-Bu

H 32 t-Bu

H 33 t-Bu

H 34 t-Bu

H 35 t-Bu

H 36 t-Bu

H 37 t-Bu

H 38 t-Bu

H 39 t-Bu

H 40 t-Bu

H 41 t-Bu

H 42 t-Bu

H 43 t-Bu

H 44 t-Bu

H 45 t-Bu

H 46 t-Bu

H 47 t-Bu

H 48 t-Bu

H 49 t-Bu

H 50 t-Bu

H 51 t-Bu

H 52 t-Bu

H 53 t-Bu

H 54 t-Bu

H 55 t-Bu

H 56 t-Bu

H 57 t-Bu

H 58 t-Bu

H 59 t-Bu

H 60 t-Bu

H 61 t-Bu

H 62 t-Bu

H 63 t-Bu

H 64 t-Bu

H 65 t-Bu

H 66 t-Bu

H 67 t-Bu

H 68 t-Bu

H 69 t-Bu

H 70 t-Bu

H 71 t-Bu

H 72 t-Bu

H 73 t-Bu

H 74 t-Bu

H 75 t-Bu

H 76 t-Bu

H 77 t-Bu

H 78 t-Bu

H 79 t-Bu

H 80 t-Bu

H 81 t-Bu

H 82 t-Bu

H 83 t-Bu

H 84 t-Bu

H 85 t-Bu

H 86 t-Bu

H 87 t-Bu

H 88 t-Bu

H 89 t-Bu

H 90 t-Bu

H 91 t-Bu

H 92 t-Bu

H 93 t-Bu

H 94 t-Bu

H 95 t-Bu

H 96 t-Bu

H 97 t-Bu

H 98 t-Bu

H 99 t-Bu

H 100 t-Bu

H 101 t-Bu

H 102 t-Bu

H 103 t-Bu

H 104 t-Bu

H 105 t-Bu

H 106 t-Bu

H 107 t-Bu

H 108 t-Bu

H 109 t-Bu

H 110 t-Bu

H 111 t-Bu

H 112 t-Bu

H 113 t-Bu

H 114 t-Bu

H 115 t-Bu

H 116 t-Bu

H 117 t-Bu

H 118 t-Bu

H 119 t-Bu

H 120 t-Bu

H 121 t-Bu

H 122 t-Bu

H 123 t-Bu

H 124 t-Bu

H 125 t-Bu

H 126 t-Bu

H 127 t-Bu

H 128 t-Bu

H 129 t-Bu

H 130 t-Bu

H 131 t-Bu

H 132 t-Bu

H 133 t-Bu

H 134 t-Bu

H 135 t-Bu

H 136 t-Bu

H 137 t-Bu

H 138 t-Bu

H 139 t-Bu

H 140 t-Bu

H 141 t-Bu

H 142 t-Bu

H 143 t-Bu

H 144 t-Bu

H

Example 3

[0055] The following cyanoenamine compounds have been tested for pesticidal activity, according to the following procedures.

[0056]Spodoptera littoralis (abbreviated as SPODLI) (commonly known as Egyptian cotton leafworm): larvicide, feeding/contact activity. Cotton leaf discs were placed on agar in petri dishes and individually sprayed with test solution of cyanoenamine in an application chamber. After drying, the leaf discs were infested with 20 to 25 L1 larvae. The samples were checked for mortality, repellent effect, feeding behavior, and growth regulation 2 and 6 days after treatment.

[0057]Heliothis virescens (abbreviated as Hv) (commonly known as tobacco budworm): ovo-larvicide, feeding/contact activity. 30 to 35 fresh eggs (0 to 24 hours old), deposited on filter paper, were placed in petri dishes on a layer of artificial diet and 0.8 mL of each test solution of cyanoenamine was individually pipetted onto them. After an incubation period of 6 days, samples were checked for egg mortality, larval mortality, and growth regulation.

[0058] Each of Spodopertera littoralis and Heliothis virescens is a larval form of an insect in the order Lepidoptera.

[0059] The results are summarized in Table B below. TABLE B Insecticidal activity (EC80 ppm) COMPOUND # SPODLI Hv

50 5

200 200

>50 >50

>>100 >100

50 50

100 100

200 200

>>100 >>100

Example 4

[0060] Construction of Reporter Plasmid

[0061] A minimal promoter vector was made by ligating a synthetic TATA box sequence oligonucleotide pair, 5′-agcttgagggtataatg-3′ (SEQ ID NO: 1) and 3′-actcccatattactcga-5′ (SEQ ID NO:2), into the HindIII site of vector pGL3-basic (Promega) so that the HindIII site was recreated 5′ to the inserted oligonucleotide and destroyed between the oligonucleotide and the downstream luciferase gene. This vector was designated TATA5.

[0062] The binding site from the hsp27 gene (Koelle et al., Cell 67(1): 59-77 (1991)) was made with the oligonucleotide pair, 5′-gatccgagacaagggttcaatgcacttgtccaatga-3′ (SEQ ID NO:3) and 3′-gctctgttcccaagttacgtgaacaggttactctag-5′ (SEQ ID NO:4). This site was multimerized and ligated into the BglII site of vector TATA-5. One isolate, pCGS154, contained the sequence below in the inserted region, having 2 pairs of sites in inverted orientations. One site had a deletion of a single base from the consensus sequence. The sequence of the inserted region in pCGS154 is shown below: 1 gatccgagac aagggttcaa tgcacttgtc caatgagatc(SEQ ID NO:5) 41 cgagacaagg gttcaatgca cttgtccaat gagatctcat 81 tggacaagtg cattgaacct tgtctcggat ctcattggac 121 aagtgcattg aacccttgtc tcggatc

[0063] Cloning of EcR Receptor Plasmid

[0064] PCR primers were designed based on the published sequence for Manduca Sexta ecdysone receptor (EcR) (genbank accession number U19812 (SEQ ID Nos:6 and 7) to clone the gene in two halves. RNA was prepared from prepupae larva of Manduca sexta using the LiCl/phenol method (Current Protocols in Molecular Biology, Vol. 1, Unit 4.3, 1987, John Wiley and Sons, publishers) and 1 μg of total RNA was used to prepare cDNA using MMLV reverse transcriptase (Promega). The cDNA was used in a PCR reaction with the primers described above to generate two PCR products for the 5′ and 3′ halves of the gene. These were subcloned into the pGEM-TA vector (Promega) and sequenced. The two fragments were joined at a unique Ndel site within each fragment and ligated into pBS-KS (Stratagene) to create a full length Manduca sexta EcR clone named pBSFLMa. A HindIII site followed by an inframe stop codon and BamHI site was placed at the 3′ end of the E domain (ligand binding domain) of the Manduca EcR receptor using the oligonucleotide: 5′-ggatcctaaagcttcgtcgtcgacacttcg-3′ (SEQ ID NO:8).

[0065] A truncated Manduca EcR containing domains C, D and E of the receptor was constructed as follows. A BamHI site and in-frame ATG was engineered just 5′ to the C domain using the degenerate primers 5′-ggatccatgggycgagaagaattrtcaccr-3′ (SEQ ID NO:9) and 5′-ccacrtcccagatctcctcga-3′ (SEQ ID NO:10). This fragment was then joined using the Nde site to the 3′ end of Manduca EcR, which has an engineered HindIII site at the 3′ end as described above.

[0066] A fragment containing the herpes simplex VP16 transactivation domain was cloned from plasmid 35S/USP-VP16 (U.S. Pat. No. 5,880,333) using the PCR primers 5′-aagcttgcccccccgaccg-3′ (SEQ ID NO:11) (placing a HindIII site at the 5′ end of the domain) and 5′-tctagaggatcctacccaccgtact-3′ (SEQ ID NO:12) (placing an inframe stop codon followed by BamHI and Xbal sites at the 3′ end of the domain). The VP16 domain was fused in frame to the 3′ end of the E domain of the ecdysone receptor using the HindIII site 3′ to EcR clone and the HindIII site engineered at the 5′ end of VP16.

[0067] The plasmid pPacU (Courey A J and Tjian R (1988) Cell 55, 887-898) was used as the starting vector for expression constructs. The truncated Manduca EcR-VP16 was ligated into pPacU using the BamHI sites flanking the coding region to create the construct referred to as MMV.

[0068] Cell-Based Assay

[0069] An in vivo cell based assay was used to measure transcriptional activation by the EcR receptor plasmid in the presence of the chemical ligands as described above. S2 Drosophila cells (ATCC CRL-1963) (commonly known as cells from the fruit fly) were transiently transfected with luciferase reporter (pCGS154) and receptor expression plasmid (MMV) using the calcium phosphate precipitation procedure (Di Nocera and David (1983) PNAS 80, 7095-7098). S2 cells were plated in 96 well format at a density of 2×10⁵ in 166.6 μl of Schneider's Drosophila media supplemented with antibiotics and 10% heat inactivated fetal bovine serum (GIBO-BRL). The following day, 33.4 μl of a calcium phosphate precipitate containing 3-6 ng of pCGS154 reporter plasmid, and 3-6 ng of EcR receptor plasmid MMV along with salmon sperm DNA, to a total of 400 ng DNA per well were added. Chemical ligands (cyanoenamine test compounds) were added 16-24 hours after DNA addition to the cells at a final concentration of 2 μM. Cells were then harvested and extracted 24 hours after chemistry addition following the procedures for the luciferase assay by centrifuging and resuspending the cell pellets in 100 μl of cell culture lysis reagent (Promega). Luciferase activity was quantitatively determined using chemiluminescence (Promega) using an analytical luminescence model 2001 luminometer. Results were normalized as a ratio of induction relative to the reporter construct without chemical ligand addition.

[0070] The results are summarized in Table C below. TABLE C Gene switch activity CHEMISTRY fold induction

66

191

225

636

1112

1145

1012

1121

29

893

239

314

364

394

913

[0071] It will be understood that various details of the invention may be changed without departing from the scope of the invention. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation—the invention being defined by the claims.

1 12 1 17 DNA Artificial Sequence primer containing synthetic TATA box 1 agcttgaggg tataatg 17 2 17 DNA Artificial Sequence primer containing synthetic TATA box 2 actcccatat tactcga 17 3 36 DNA Artificial Sequence PCR primer for amplification of Drosophila melanogaster DNA 3 gatccgagac aagggttcaa tgcacttgtc caatga 36 4 36 DNA Artificial Sequence PCR primer for amplification of Drosophila melanogaster DNA 4 gctctgttcc caagttacgt gaacaggtta ctctag 36 5 147 DNA Artificial Sequence multimerized binding site of Drosophila melanogaster hsp27 gene 5 gatccgagac aagggttcaa tgcacttgtc caatgagatc cgagacaagg gttcaatgca 60 cttgtccaat gagatctcat tggacaagtg cattgaacct tgtctcggat ctcattggac 120 aagtgcattg aacccttgtc tcggatc 147 6 2840 DNA Manduca sexta CDS (361)..(2031) Manduca sexta Ecdysone Receptor 6 tccgttgacg acggtcgcac gcgtgcaacg tgctcgtttt tacggctcaa gcgaacgcgt 60 aacctccgtc tccacatcac cgagcgaact ctagaactcg cgtactcttc tcacctgttg 120 cttcggattg tgttgtgact gaaaagcgac gcgtatcgtg gtcgaagatt ctctataagt 180 gcataatata ttcgagacag tggatagcga ttcgtttcgg tttcatcgcg cggatgagtg 240 gttcatgccc gtagagacgc gtttagatag ttatggcgag gaaaaagtga agtgaaagcc 300 tacgtcagag gatgtccctc ggtggtcacg gaagccgggg cgtgtgacgc gctcttcgac 360 atg aga cgc cgc tgg tca aac aac gga tgt ttc cct ctg cga atg ttt 408 Met Arg Arg Arg Trp Ser Asn Asn Gly Cys Phe Pro Leu Arg Met Phe 1 5 10 15 gag gag agc tcc tct gaa gtg act tct tcc tcg gcg ttc ggg atg ccg 456 Glu Glu Ser Ser Ser Glu Val Thr Ser Ser Ser Ala Phe Gly Met Pro 20 25 30 gcg gcc atg gta atg tca ccg gag tcg ctg gcg tcg cca gag tac ggc 504 Ala Ala Met Val Met Ser Pro Glu Ser Leu Ala Ser Pro Glu Tyr Gly 35 40 45 ggc ctc gag ctc tgg agc tac gat gag acc atg aca aac tat ccg gcg 552 Gly Leu Glu Leu Trp Ser Tyr Asp Glu Thr Met Thr Asn Tyr Pro Ala 50 55 60 cag tca ctg ctc ggc gcg tgt aat gcg ccg cag cag cag cag caa cag 600 Gln Ser Leu Leu Gly Ala Cys Asn Ala Pro Gln Gln Gln Gln Gln Gln 65 70 75 80 caa caa cag cag ccg tcc gct cag ccg ctg ccg tct atg ccg ctg ccg 648 Gln Gln Gln Gln Pro Ser Ala Gln Pro Leu Pro Ser Met Pro Leu Pro 85 90 95 atg cct cct aca act cct aaa tca gag aac gag tcc atg tcg tca ggt 696 Met Pro Pro Thr Thr Pro Lys Ser Glu Asn Glu Ser Met Ser Ser Gly 100 105 110 cga gaa gaa tta tca ccg gcc tca agt ata aat gga tgt agt act gat 744 Arg Glu Glu Leu Ser Pro Ala Ser Ser Ile Asn Gly Cys Ser Thr Asp 115 120 125 ggg gaa cca aga cga cag aag aaa ggg cca gcg ccg cgc cag cag gag 792 Gly Glu Pro Arg Arg Gln Lys Lys Gly Pro Ala Pro Arg Gln Gln Glu 130 135 140 gaa ctg tgc ctt gtt tgc ggc gac agg gct tcg gga tat cac tat aac 840 Glu Leu Cys Leu Val Cys Gly Asp Arg Ala Ser Gly Tyr His Tyr Asn 145 150 155 160 gcg ctt acg tgc gaa gga tgt aaa ggg ttc ttc agg cgg agt gtg acc 888 Ala Leu Thr Cys Glu Gly Cys Lys Gly Phe Phe Arg Arg Ser Val Thr 165 170 175 aag aat gcg gta tat att tgt aaa ttt gga cac gcc tgc gag atg gac 936 Lys Asn Ala Val Tyr Ile Cys Lys Phe Gly His Ala Cys Glu Met Asp 180 185 190 atg tac atg agg aga aaa tgc caa gag tgt cgg ttg aag aaa tgc ctc 984 Met Tyr Met Arg Arg Lys Cys Gln Glu Cys Arg Leu Lys Lys Cys Leu 195 200 205 gcg gtg ggc atg agg ccc gag tgc gtc gtc cca gag tcc acg tgc aag 1032 Ala Val Gly Met Arg Pro Glu Cys Val Val Pro Glu Ser Thr Cys Lys 210 215 220 aac aaa aga aga gaa aag gaa gca cag aga gaa aaa gac aaa ctg cca 1080 Asn Lys Arg Arg Glu Lys Glu Ala Gln Arg Glu Lys Asp Lys Leu Pro 225 230 235 240 gtc agt acg acg aca gtg gac gat cat atg cct gcc ata atg caa tgt 1128 Val Ser Thr Thr Thr Val Asp Asp His Met Pro Ala Ile Met Gln Cys 245 250 255 gac cct ccg ccc cca gag gcg gca agg att cac gaa gtg gtc ccg agg 1176 Asp Pro Pro Pro Pro Glu Ala Ala Arg Ile His Glu Val Val Pro Arg 260 265 270 ttc cta acg gag aag cta atg gag cag aac aga ctg aag aat gtg acg 1224 Phe Leu Thr Glu Lys Leu Met Glu Gln Asn Arg Leu Lys Asn Val Thr 275 280 285 ccg ctg tcg gcg aac cag aag tcc ctg atc gcg agg ctc gtg tgg tac 1272 Pro Leu Ser Ala Asn Gln Lys Ser Leu Ile Ala Arg Leu Val Trp Tyr 290 295 300 cag gag ggg tac gag cag ccg tcg gag gaa gat ctc aag aga gtt aca 1320 Gln Glu Gly Tyr Glu Gln Pro Ser Glu Glu Asp Leu Lys Arg Val Thr 305 310 315 320 cag aca tgg cag tta gaa gaa gaa gaa gag gag gaa act gac atg ccc 1368 Gln Thr Trp Gln Leu Glu Glu Glu Glu Glu Glu Glu Thr Asp Met Pro 325 330 335 ttc cgt cag atc aca gag atg acg atc tta aca gtg cag ctt att gta 1416 Phe Arg Gln Ile Thr Glu Met Thr Ile Leu Thr Val Gln Leu Ile Val 340 345 350 gaa ttc gca aag gga cta ccg gga ttc tcc aag ata tct cag tcc gat 1464 Glu Phe Ala Lys Gly Leu Pro Gly Phe Ser Lys Ile Ser Gln Ser Asp 355 360 365 caa att aca tta tta aag gcg tca tca agc gaa gtg atg atg ctg cga 1512 Gln Ile Thr Leu Leu Lys Ala Ser Ser Ser Glu Val Met Met Leu Arg 370 375 380 gtg gcg cga cgg tac gac gcg gcg acg gac agc gtg ctg ttc gcg aac 1560 Val Ala Arg Arg Tyr Asp Ala Ala Thr Asp Ser Val Leu Phe Ala Asn 385 390 395 400 aac cag gcg tac acg cgc gac aac tac cgc aag gcg ggc atg tcc tac 1608 Asn Gln Ala Tyr Thr Arg Asp Asn Tyr Arg Lys Ala Gly Met Ser Tyr 405 410 415 gtc atc gag gac ctg ctg cac ttc tgt cgg tgt atg tac tcc atg agc 1656 Val Ile Glu Asp Leu Leu His Phe Cys Arg Cys Met Tyr Ser Met Ser 420 425 430 atg gac aat gtg cac tac gcg ctg ctc acc gcc atc gtt ata ttc tca 1704 Met Asp Asn Val His Tyr Ala Leu Leu Thr Ala Ile Val Ile Phe Ser 435 440 445 gac cgg cca ggc ctc gag caa ccc ctt tta gtg gag gaa atc cag aga 1752 Asp Arg Pro Gly Leu Glu Gln Pro Leu Leu Val Glu Glu Ile Gln Arg 450 455 460 tac tac ttg aag acg ctg cgg gtt tac att tta aat cag cac agc gcg 1800 Tyr Tyr Leu Lys Thr Leu Arg Val Tyr Ile Leu Asn Gln His Ser Ala 465 470 475 480 tcg cct cgc tgc gcc gtg ctg ttc ggc aag atc ctc ggc gtg ctg acg 1848 Ser Pro Arg Cys Ala Val Leu Phe Gly Lys Ile Leu Gly Val Leu Thr 485 490 495 gaa ctg cgc acg ctc ggc acg cag aac tcc aac atg tgc atc tcg ctg 1896 Glu Leu Arg Thr Leu Gly Thr Gln Asn Ser Asn Met Cys Ile Ser Leu 500 505 510 aag ctg aag aac agg aaa ctt ccg cca ttc ctc gag gag atc tgg gac 1944 Lys Leu Lys Asn Arg Lys Leu Pro Pro Phe Leu Glu Glu Ile Trp Asp 515 520 525 gtg gcc gaa gtg tcg acg acg cag ccg acg ccg ggg gtg gcg gcg cag 1992 Val Ala Glu Val Ser Thr Thr Gln Pro Thr Pro Gly Val Ala Ala Gln 530 535 540 gtg acc ccc atc gtg gtg gac aac ccc gcg gcg ctc tag ctggcgcgcc 2041 Val Thr Pro Ile Val Val Asp Asn Pro Ala Ala Leu 545 550 555 ggcgccgcgc cccgccgccc ccgccgccgc cgctcccccg cgccgccgcc gcgcgccccc 2101 gcggcctgcg ctgagtgcgg gacccgcccc gaggagagaa cgctcataga ctggctagtt 2161 ttagtgaagt gcacggacgc gatcgtggga ccgcatcgac gcgtccgtga ggacagtgca 2221 aatattaccg ctagggccgg ttcgtacgtg tccggtgacc gacgacgatg atgcgcgtga 2281 gattagtgaa tatatgtgtt gttgaacgtt tggagagtat atttagtgtt gatcgtcggg 2341 agcgcgcggc cggcgcgtgt cggcgagctg tccgccgcgc gccggccgcg gcgactccgc 2401 gtttttttcg tttgcgaccg gaaaccgagt cggtcactcg gatacgcccg tatgataaga 2461 cttctttcga taaataagtt cacctgtatt gcgcgtacat acgagaatta taaagaaaaa 2521 aagtaatata tgaagagatg tttctattgg gtgaaaagtt taaacttatg tttatttacc 2581 aaaattaact atacgttgat cgaccttttg actataatat tgtgctgggt cgttggcagc 2641 ggccgacgaa cgcgcgccga ccatatttgt ttatatatag tttatgtgag acgttatcgt 2701 gtcgtgtcca cttagttccg attcatgttc caccaggtcg gtgtagtgat cagggcgggc 2761 cagggtgacg gccaccacgg ataacaggca aagagcgacg aatgttttca tgttgagact 2821 ttgggagacg ttattcctc 2840 7 556 PRT Manduca sexta 7 Met Arg Arg Arg Trp Ser Asn Asn Gly Cys Phe Pro Leu Arg Met Phe 1 5 10 15 Glu Glu Ser Ser Ser Glu Val Thr Ser Ser Ser Ala Phe Gly Met Pro 20 25 30 Ala Ala Met Val Met Ser Pro Glu Ser Leu Ala Ser Pro Glu Tyr Gly 35 40 45 Gly Leu Glu Leu Trp Ser Tyr Asp Glu Thr Met Thr Asn Tyr Pro Ala 50 55 60 Gln Ser Leu Leu Gly Ala Cys Asn Ala Pro Gln Gln Gln Gln Gln Gln 65 70 75 80 Gln Gln Gln Gln Pro Ser Ala Gln Pro Leu Pro Ser Met Pro Leu Pro 85 90 95 Met Pro Pro Thr Thr Pro Lys Ser Glu Asn Glu Ser Met Ser Ser Gly 100 105 110 Arg Glu Glu Leu Ser Pro Ala Ser Ser Ile Asn Gly Cys Ser Thr Asp 115 120 125 Gly Glu Pro Arg Arg Gln Lys Lys Gly Pro Ala Pro Arg Gln Gln Glu 130 135 140 Glu Leu Cys Leu Val Cys Gly Asp Arg Ala Ser Gly Tyr His Tyr Asn 145 150 155 160 Ala Leu Thr Cys Glu Gly Cys Lys Gly Phe Phe Arg Arg Ser Val Thr 165 170 175 Lys Asn Ala Val Tyr Ile Cys Lys Phe Gly His Ala Cys Glu Met Asp 180 185 190 Met Tyr Met Arg Arg Lys Cys Gln Glu Cys Arg Leu Lys Lys Cys Leu 195 200 205 Ala Val Gly Met Arg Pro Glu Cys Val Val Pro Glu Ser Thr Cys Lys 210 215 220 Asn Lys Arg Arg Glu Lys Glu Ala Gln Arg Glu Lys Asp Lys Leu Pro 225 230 235 240 Val Ser Thr Thr Thr Val Asp Asp His Met Pro Ala Ile Met Gln Cys 245 250 255 Asp Pro Pro Pro Pro Glu Ala Ala Arg Ile His Glu Val Val Pro Arg 260 265 270 Phe Leu Thr Glu Lys Leu Met Glu Gln Asn Arg Leu Lys Asn Val Thr 275 280 285 Pro Leu Ser Ala Asn Gln Lys Ser Leu Ile Ala Arg Leu Val Trp Tyr 290 295 300 Gln Glu Gly Tyr Glu Gln Pro Ser Glu Glu Asp Leu Lys Arg Val Thr 305 310 315 320 Gln Thr Trp Gln Leu Glu Glu Glu Glu Glu Glu Glu Thr Asp Met Pro 325 330 335 Phe Arg Gln Ile Thr Glu Met Thr Ile Leu Thr Val Gln Leu Ile Val 340 345 350 Glu Phe Ala Lys Gly Leu Pro Gly Phe Ser Lys Ile Ser Gln Ser Asp 355 360 365 Gln Ile Thr Leu Leu Lys Ala Ser Ser Ser Glu Val Met Met Leu Arg 370 375 380 Val Ala Arg Arg Tyr Asp Ala Ala Thr Asp Ser Val Leu Phe Ala Asn 385 390 395 400 Asn Gln Ala Tyr Thr Arg Asp Asn Tyr Arg Lys Ala Gly Met Ser Tyr 405 410 415 Val Ile Glu Asp Leu Leu His Phe Cys Arg Cys Met Tyr Ser Met Ser 420 425 430 Met Asp Asn Val His Tyr Ala Leu Leu Thr Ala Ile Val Ile Phe Ser 435 440 445 Asp Arg Pro Gly Leu Glu Gln Pro Leu Leu Val Glu Glu Ile Gln Arg 450 455 460 Tyr Tyr Leu Lys Thr Leu Arg Val Tyr Ile Leu Asn Gln His Ser Ala 465 470 475 480 Ser Pro Arg Cys Ala Val Leu Phe Gly Lys Ile Leu Gly Val Leu Thr 485 490 495 Glu Leu Arg Thr Leu Gly Thr Gln Asn Ser Asn Met Cys Ile Ser Leu 500 505 510 Lys Leu Lys Asn Arg Lys Leu Pro Pro Phe Leu Glu Glu Ile Trp Asp 515 520 525 Val Ala Glu Val Ser Thr Thr Gln Pro Thr Pro Gly Val Ala Ala Gln 530 535 540 Val Thr Pro Ile Val Val Asp Asn Pro Ala Ala Leu 545 550 555 8 30 DNA Artificial Sequence PCR primer for amplification of Manduca sexta DNA 8 ggatcctaaa gcttcgtcgt cgacacttcg 30 9 30 DNA Artificial Sequence PCR primer for amplification of Manduca sexta DNA 9 ggatccatgg gycgagaaga attrtcaccr 30 10 21 DNA Artificial Sequence PCR primer for amplification of Manduca sexta DNA 10 ccacrtccca gatctcctcg a 21 11 19 DNA Artificial Sequence PCR primer for amplification of Manduca sexta DNA 11 aagcttgccc ccccgaccg 19 12 25 DNA Artificial Sequence PCR primer for amplification of Manduca sexta DNA 12 tctagaggat cctacccacc gtact 25 

What is claimed is:
 1. A compound comprising Formula I

wherein: R1 is a branched chain C3 to C8 alkyl, C3 to C8 cycloalkyl, C4 to C8 alkyl-substituted alkyl, bicycloalkyl, 1-adamantyl, polyhaloalkyl, trialkylsilyl, unsubstituted phenyl, or substituted phenyl; R2 and R3 are each independently of the other unsubstituted or substituted aromatic rings, chosen from phenyl, pyridyl, pyrimidinyl, furyl, thiophenyl, pyrazinyl, pyrrolyl, pyrazolyl, 1,2,4-triazolyl, naphthyl, fluorenonyl, xanthenyl, 4-oxo-1,4-dihydro-(1,8)naphthyridinyl, thiazolyl, isothiazolyl, 1,3,4-thiadiazolyl, benzo-1,2,3-thiadiazolyl, oxazolyl, imidazolyl, quinolinyl, or isoquinolinyl, where a substituent on the rings is one or more chosen independently from hydrogen, C1 to C4 alkyl, alkoxy, alkoxyalkyl, hydroxy, amino, alkylamino, dialkylamino, acylamino, halo, haloalkyl, hydroxyalkyl, dihydroxyalkyl, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, unsubstituted or substituted alkylphenyl, unsubstituted or substituted phenyl, unsubstituted or substituted phenoxy, nitro, cyano, alkylthio, alkylsulfonyl, aminoalkyl, carboxyalkyl, or sulfonylalkyl; and R4 is hydrogen, alkylthio, alkylthioalkyl, alkyloxyalkyl, acyloxyalkyl, alkyl, acyl, trialkylsilyl, or is taken together with R3 and the O in Formula I to form a lactone ring; and the salts, stereoisomers, and tautomers thereof.
 2. The compound of claim 1, wherein R1 is tert-butyl.
 3. The compound of claim 1, wherein at least one of R2 and R3 is substituted with a substituent forming a cyclic structure on adjacent atoms of the aromatic ring.
 4. The compound of claim 3, wherein the substituent is selected from the group consisting of 1,2-methylenedioxy and 1,2-difluoromethylenedioxy.
 5. The compound of claim 1, wherein R2 is selected from the group consisting of phenyl, 3,5-dimethylphenyl, 2,4-dimethylphenyl, 3-methylphenyl, 4-methylphenyl, 2-methylphenyl, and 3,4-methoxydioxyphenyl.
 6. The compound of claim 1, wherein R3 is selected from the group consisting of phenyl, 3-pyridyl, 3-methoxy-2-methylphenyl, 3-ethoxy-2-methylphenyl, 3-methoxy-2-ethylphenyl, 4-ethylphenyl, 2,6-difluorophenyl, 2,3-dimethylphenyl, 3-chloro-2-methylphenyl, and 3-bromo-2-methylphenyl.
 7. The compound of claim 1, wherein halo is selected from the group consisting of fluoro, chloro, bromo, iodo, and combinations thereof.
 8. The compound of claim 1, wherein Formula I is in its tautomeric form as Formula II:


9. The tautomeric compound of claim 8, wherein R3 and R4 and O together form a cyclic structure resulting in a lactone.
 10. The compound of claim 9, wherein the lactone is selected from the group consisting of:


11. The compound of claim 1, wherein Formula I is in its isomeric form as Formula III:


12. The isomeric compound of claim 11, wherein R1 is tert-butyl, R2 is 3,5-dimethylphenyl, and R3 is fluoromethylphenyl or 2-methyl-3-methoxyphenyl.
 13. The isomeric compound of claim 12, wherein the compound is selected from the group consisting of:


14. The compound of claim 1, wherein the compound is selected from the group consisting of:


15. A method of controlling gene expression comprising contacting an ecdysone receptor gene switch with a compound of Formula I

wherein: R1 is a branched chain C3 to C8 alkyl, C3 to C8 cycloalkyl, C4 to C8 alkyl-substituted alkyl, bicycloalkyl, 1-adamantyl, polyhaloalkyl, trialkylsilyl, unsubstituted phenyl, or substituted phenyl; R2 and R3 are each independently of the other unsubstituted or substituted aromatic rings, chosen from phenyl, pyridyl, pyrimidinyl, furyl, thiophenyl, pyrazinyl, pyrrolyl, pyrazolyl, 1,2,4-triazolyl, naphthyl, fluorenonyl, xanthenyl, 4-oxo-1,4-dihydro-(1,8)naphthyridinyl, thiazolyl, isothiazolyl, 1,3,4-thiadiazolyl, benzo-1,2,3-thiadiazolyl, oxazolyl, imidazolyl, quinolinyl, or isoquinolinyl, where a substituent on the rings is one or more chosen independently from hydrogen, C1 to C4 alkyl, alkoxy, alkoxyalkyl, hydroxy, amino, alkylamino, dialkylamino, acylamino, halo, haloalkyl, hydroxyalkyl, dihydroxyalkyl, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, unsubstituted or substituted alkylphenyl, unsubstituted or substituted phenyl, unsubstituted or substituted phenoxy, nitro, cyano, alkylthio, alkylsulfonyl, aminoalkyl, carboxyalkyl, or sulfonylalkyl; and R4 is hydrogen, alkylthio, alkylthioalkyl, alkyloxyalkyl, acyloxyalkyl, alkyl, acyl, trialkylsilyl, or is taken together with R3 and the O in Formula I to form a lactone ring; and the salts, stereoisomers, and tautomers thereof.
 16. The method of claim 15, wherein R1 is tert-butyl.
 17. The method of claim 15, wherein at least one of R2 and R3 is substituted with a substituent forming a cyclic structure on adjacent atoms of the aromatic ring.
 18. The method of claim 17, wherein the substituent is selected from the group consisting of 1,2-methylenedioxy and 1,2-difluoromethylenedioxy.
 19. The method of claim 15, wherein R2 is selected from the group consisting of phenyl, 3,5-dimethylphenyl, 2,4-dimethylphenyl, 3-methylphenyl, 4-methylphenyl, 2-methylphenyl, and 3,4-methoxydioxyphenyl.
 20. The method of claim 15, wherein R3 is selected from the group consisting of phenyl, 3-pyridyl, 3-methoxy-2-methylphenyl, 3-ethoxy-2-methylphenyl, 3-methoxy-2-ethylphenyl, 4-ethylphenyl, 2,6-difluorophenyl, 2,3-dimethylphenyl, 3-chloro-2-methylphenyl, and 3-bromo-2-methylphenyl.
 21. The method of claim 15, wherein halo is selected from the group consisting of fluoro, chloro, bromo, iodo, and combinations thereof.
 22. The method of claim 15, wherein Formula I is in its tautomeric form as Formula II:


23. The method of claim 22, wherein in the tautomeric form, R3 and R4 and O together form a cyclic structure resulting in a lactone.
 24. The method of claim 23, wherein the lactone is selected from the group consisting of:


25. The method of claim 15, wherein Formula I is in its isomeric form as Formula III:


26. The isomeric method of claim 25, wherein R1 is tert-butyl, R2 is 3,5-dimethylphenyl, and R3 is 2-trifluoromethylphenyl or 2-methyl-3-methoxyphenyl.
 27. The isomeric method of claim 26, wherein the compound is selected from the group consisting of:


28. The method of claim 15, wherein the compound is selected from the group consisting of: 